The present invention generally relates to the field of therapeutic and diagnostic nucleic acid preparatives, as a novel Hoogsteen duplex structure that can be used to selectively hybridize with a target third RNA or DNA strand by Watson-Crick pairing, thus forming a triplex, is disclosed. The present invention also relates to the field of xe2x80x9cantisensexe2x80x9d therapy, as a method whereby nucleic acid structures are used to hybridize with mRNA and control gene expression through inhibiting translation, is provided. The invention further relates to the use of xe2x80x9cantigenexe2x80x9d nucleic acid structures, as these structures are used by the present inventors to hybridize with a strand of DNA and control gene expression by inhibiting transcription.
Antisense and antigene control of gene expression are two ol the more promising new biotechnology techniques (1-6). In general, the antisense technology involves the binding of one strand of nucleic acid, which may be normal or modified DNA or RNA, to a target single-stranded sequence in mRNA to form a duplex. The antigene technology generally involves the binding of one strand of nucleic acid, which may be normal or modified DNA or RNA, to a target sequence in the double-stranded DNA genome to form a triplex.
The two major nucleic acid triple-helix motifs with Hoogsteen or reverse-Hoogsteen pairing of a third strand to a DNA duplex, and the numerous ways in which a third strand might inhibit transcription or mediate mutagenesis and recombination in antigene drug therapies, have been reviewed (4-6). There is some evidence that suggests triplexes may form under in vivo conditions (7-14), and may involve RNA (15). With RNA as one or more of the strands, triple helix formation could potentially be used to control biological processes that encompass mRNA, RNA:DNA hybrids, or RNA hairpins.
There are eight possible combinations of DNA and RNA strands in a triplex. Roberts and Crothers (18) explored the effects of backbone composition on pyr*pur:pyr triplex stability using four Watson-Crick-paired hairpin duplexes (D:D, D:R, R:D, and R:R) having 12 base pairs, including 8 G:C pairs, in the stems. These were mixed with homopyrdimidine DNA (D) or RNA (R) 12-mers of the proper sequences to form Hoogsteen pairs. At pH 5.5, the D*R:D and D*R:R triplexes did not form. In general, a triplex was formed with a Hoogsteen DNA pyrimidine strand only when it could pair with a DNA purine strand. Melting was by Scheme I or II of FIG. 1. Four triplexes (D*D:D, D*D:R, R*R:D, and R*R:R) melted with two transitions, with the lower Tm values corresponding to the separation of the third Hoogsteen-paired strand. Only one, or two very close, transitions were found for the R*D:D and R*D:R triplexes, indicating that the Hoogsteen-paired strand was stable until the hairpin duplex melted. Noronha and Damha (19, 20) confirmed and extended the Roberts and Crothers (18) results with the same hairpin duplex sequences. Other workers used relatively long Watson-Crick duplexes to make partial triplexes with short Hoogsteen-paired third strands (21-23). Again it was found that the D*R:D and D*R:R triplexes did not form, R*D:D and R*D:R were among the most stable triplexes, and the shorter Hoogsteen-paired oligomers always melted at a lower temperature than the Watson-Crick duplexes.
Many studies of triplexes, including NMR and thermodynamic studies, have made use of constructs in which two or all three strands are covalently linked (24-36). The relative stability of the Hoogsteen-paired strand is more certain when mixtures are made of separated oligomers. Liu et al. (37) recognized that unlinked strands can form a Hoogsteen-paired DNA duplex at pH 5.1. The strand sequences were constructed so that an antiparallel Watson-Crick alignment would not be stable. Whether Watson-Crick or Hoogsteen duplexes are more stable in mixtures of separate strands depends on the sequences and conditions involved (17, 18, 38). Lavelle and Fresco (17) inferred from optical and thermodynamic studies that the two melting transitions of a d(C+T)6*d(AG)6:d(CT)6 (SEQ ID NO: 15) triplex formed from separate strands at pH 4.2 represented (a) a dissociation at lower temperatures of the Watson-Crick pair and (b) the dissociation of a more stable Hoogsteen-paired duplex at higher temperatures. That is, dissociation occurred by Scheme III of FIG. 1. At higher pH values, the Hoogsteen-paired strand was less stable and dissociated first.
In some aspects, the present invention provides a method of users a preformed double-stranded nucleic acid structure consisting of two parallel Hoogsteen-base-paired strands to bind to a single-stranded target in mRNA or one strand of the double-stranded DNA genome. In both cases, the resulting structure is a triplex.
In the pyrimidine*purine:pyrimidine (pyr*pur:pyr) motif, the subject of this application, the strands are restricted to being all homopurine or homopyrimidine. xe2x80x9c*xe2x80x9d and xe2x80x9c:xe2x80x9d respectively denote pairing between parallel Hoogsteen-paired and antiparallel Watson-Crick-paired strands. The pyr*pur:pyr triplex contains protonated C+*G:C base triples, diagrammed at the top of FIG. 1. The G forms 3 hydrogen bonds with one C by Watson-Crick pairing and 2 hydrogen bonds with a second C+ by Hoogsteen pairing. The cytosines of a Hoogsteen pair are protonated at their N3 positions, and the triplex preferentially forms at low pH. However, the pKa for protonation of a hydrogen-bonded cytosine N3 can be well above the pKa of 4.6 for isolated cytidylic acid. The requirement for protonation of Hoogsteen-paired C+ bases leads to enhanced triplex stability at low pH (16,17). It is also possible to form unprotonated T*A:T or U*A:U base triples that are isostructural with the C+*G:C base triple. Thus, these triple helices have all-pyrimidine and all-purine strands arranged as pyr*pur:pyr.
The present inventors have discovered that R*D:R as well as D*D:D triplexes formed from separate d(AG)12 and d(CT)12 or r(CU)12 strands at low pH melt with two transitions by Scheme III of FIG. 1. Thus, the Watson-Crick strand can be preferentially dissociated by increasing the temperature at low pH, resulting in a stable structure consisting of only the Hoogsteen-paired duplex even when the strands are not covalently linked. The Hoogsteen duplex can be an RNA*DNA hybrid or a DNA*DNA duplex. The preferred Hoogsteen duplex is the RNA*DNA hybrid. Covalent linkage of the two strands would provide even greater stability.
The solution constraints and conditions for forming and isolating Hoogsteen-paired duplexes is described in the context of the present invention. A Hoogsteen-paired RNA*DNA hybrid duplex that can bind to a third RNA strand with high affinity is used in one aspect of the present invention as part of a new type of enhanced therapeutic antisense molecule. A Hoogsteen-paired RNA*DNA duplex can bind to a third DNA strand with high affinity, predicted to be higher than that of binding of a DNA*DNA duplex to a third DNA strand, and thus may be used as a new type of enhanced therapeutic antigene molecule.
Antisense DNA drug therapy (39) has been shown in at least some instances to be an effective means of controlling disease processes at the translational level of gene expression. At least 12 clinical trials using antisense methods are underway (1), and progress has been made in treating HIV infection, xcex2-globin thalassemia, stenosis (constriction) of artery-replaced vein grafts, diabetic retinopathy, various cancers, hematological malignancies, malaria, hepatitis B infection, papillomavirus genital warts, and retinitis caused by cytomegalovirus (1, 2). The antisense effect is typically obtained by hybridization of a DNA oligonucleotide (typically about 20 nucleotides long) to a complementary sequence within an mRNA. The hybrid then physically blocks translation or activates RNase H degradation of the mRNA.
Most of the first generation of antisense drugs consist of phosphorothioate DNAs (S-DNAs), in which sulfur replaces one of the phosphodioxy oxygens in each nucleotide subunit of the oligonucleotide. One disadvantage of S-DNAs is that the phosphorothioate substitution reduces the stability of S-DNA:RNA hybrids relative to hybrids with unmodified DNAs (40). A second generation of antisense drugs has base, sugar, and phosphate modifications that enhance hybrid stability. Another strategy to increase the antisense effect is to combine different modifications in a xe2x80x9cgapmerxe2x80x9d DNA that has other nucleotide modifications flanking an S-DNA segment, which is retained since S-DNA:RNA hybrids activate RNase H (2, 41). General problems that must be solved on a case-by-case basis are that some antisense DNA sequences form competing self-complexes such as hairpins or may bind non-specifically to cellular proteins.
In one aspect the present invention provides a multistranded, or xe2x80x9chyperstructuredxe2x80x9d, antisense molecule. This molecule consists of two parallel-stranded Hoogsteen-paired strands that, compared with single-stranded S-DNAs, are less likely to form self-complexes and would be able to form a more stable complex with a target mRNA by triplex pairing. Triplex formation could either physically block translation, as illustrated in FIG. 2, or be attached to an S-DNA segment to activate RNase H.
The ability of a single stranded DNA or RNA to form a triplex structure is the basis of an xe2x80x9cantigenexe2x80x9d drug. The result is to control transcription, where the DNA duplex gene is the target (4-6, 39). This use of triplexes differs from that described in the present invention to control transcription. In some embodiments of the invention, the target is one of the single-strands of the duplex, and the antigene molecule is itself a duplex that has Hoogsteen base pairs.
Foldback triplex-forming oligonucleotides (FTFOs) (Kandimalla et al., U.S. Pat. No. 5,693,773 (1997)) consist of a continuous strand with all the nucleotides linked in one 5xe2x80x2 to 3xe2x80x2 direction. FTFOs fold so that there is an antiparallel arrangement of typically two pyrimidine domains of this molecule that both directly pair with the target. One domain pairs parallel and one domain pairs antiparallel to the target sequence direction. Folding is simultaneous upon forming a triplex.
The stem-loop oligonucleotides (Kool, U.S. Pat. No. 5,808,036 (1998) consist of a loop region with nucleotides linked in one 5xe2x80x2 to 3xe2x80x2 direction. Different domains of the loop are like domains of FTFOs in that they typically are pyrimidine rich and both directly pair with the target. Again, one domain pairs parallel and one domain pairs antiparallel to the target sequence direction, and the domains of the loop are not in a duplex form before adding the target nucleic acid.
In other embodiments, the invention comprises a nucleic acid composition comprising two strands or two domains that are parallel and paired prior to forming a triplex. In particular embodiments, the invention provides one pyridimine strand or domain and one purine strand or domain, with any linkage being such that the strands are linked head-to-head or tail-to-tail and all the nucleotides are not in the same 5xe2x80x2 to 3xe2x80x2 direction.